- Neovastat(Æ-941) inhibits the airway inflammation and hyperresponsiveness in a murine model of asthma
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Sook-Young Lee , Soon-Young Paik , Su-Mi Chung ,
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J. Microbiol. 2005;43(1):11-16.
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DOI: https://doi.org/2145 [pii]
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Matrix metalloproteinase (MMP)-9 plays an important role in the pathogenesis of bronchial asthma. Neovastat, having significant antitumor and antimetastatic properties, is classified as a naturally occurring multifunctional antiangiogenic agent. We evaluated the therapeutic effect of Neovastat on airway inflammation in a mouse model of asthma. BALB/c mice were immunized subcutaneously with ovalbumin (OVA) on days 0, 7, 14, and 21 and challenged with inhaled OVA on days 26, 29, and 31. Neovastat was administrated by gavage (5 mg/kg body weight) three times with 12 h intervals, beginning 30 min before OVA inhalation. On day 32, mice were challenged with inhaled methacholine, and enhanced pause (Penh) was measured as an index of airway hyperresponsiveness. The severity of airway inflammation was determined by differential cell count of bronchoalveolar lavage (BAL) fluid. The MMP-9 concentration in BAL fluid samples was measured by ELISA, and MMP-9 activity was measured by zymography. The untreated asthma group showed an increased inflammatory cell count in BAL fluid and Penh value compared with the normal control group. Mice treated with Neovastat had significantly reduced Penh values and inflammatory cell counts in BAL fluid compared with untreated asthmatic mice. Furthermore, mice treated with Neovastat showed significantly reduced MMP-9 concentrations and activity in BAL fluid. These results demonstrate that Neovastat might have new therapeutic potential for airway asthmatic inflammation.
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- PCR Method Based on the ogdH Gene for the Detection of Salmonella spp. from Chicken Meat Samples
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Un-Ho Jin , Sung-Hak Cho , Min-Gon Kim , Sang-Do Ha , Keun-Sung Kim , Kyu-Ho Lee , Kwang-Yup Kim , Duck Hwa Chung , Young-Choon Lee , Cheorl-Ho Kim
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J. Microbiol. 2004;42(3):216-222.
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DOI: https://doi.org/2086 [pii]
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In a previous paper, the ogdH gene that encodes 2-oxoglutarate dehydrogenase was isolated from Salmonella typhimurium. The catalytic N-terminal region in the enzyme was found to be very specific for the Salmonella species. Therefore, the aim of the present study was to detect S. typhimurium in food sources using primers designed for OGDH-1 and OGDH-2 which were based on the salmonella-specific region of the ogdH gene. A simple polymerase chain reaction (PCR) detection method was developed to detect low numbers of S. typhimurium in a chicken meat microbial consortium. Using the ogdH-specific primers under stringent amplification conditions and for gene probe analysis, fewer than 100 colony-forming units (CFUs) were detectable when pure cultures were employed. When the PCR assay was run on S. typhimurium-contaminated meat contents, only the positive meat samples containing as few as 200 CFUs reacted to the assay. The method employed for sample processing is simple and it was determined to provide a sensitive means of detecting trace amounts of S. typhimurium-specific sequences in the presence of mixed meat microbial populations. When compared with six representative intestinal gram-negative bacterial strains in foods, including Vibrio parahaemolyticus, V. vulnificus, Enterobacter cloacae, E. coli O157:H7, Pseudomonas aeruginosa, and Proteus sp., S. typhimurium had a unique and distinct PCR product (796 bp). In conclusion, the two OGDH primers were found to be rapid and sensitive detectors of Salmonella spp for the PCR method.
- Impact of Genetically Modified Enterobacter cloacae on Indigenous Endophytic Community of Citrus sinensis Seedlings
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Fernando Dini Andreote , Marcelo Jose Mortatti Gullo , Andre Oliveira de Souza Lima , Walter Maccheroni Junior , Joao Lucio Azevedo , Welington Luiz Araujo
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J. Microbiol. 2004;42(3):169-173.
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Enterobacter cloacae (strain PR2/7), a genetically modified endophyte (GME) in citrus plants, carrying different plasmids (pEC3.0/18, pCelE, pEglA and pGFP), was inoculated into Citrus sinensis seedlings under greenhouse conditions. The impact of this on the indigenous bacterial endophytic community was studied by analyses of 2 different morphologic groups. The germination rates of inoculated seeds were evaluated in greenhouse, and plasmid stability under in vitro conditions. Results demonstrated a great and diverse endophytic community inside plants, and specialization in tissue colonization by some bacterial groups, in different treatments. Shifts in seed germination rate were observed among treatments: in general, the PR2/7 harboring pEglA bacterial clone significantly reduced seed germination, compared to the PR2/7 harboring pEC3.0/18 clone. This suggests that the presence of the pEglA plasmid changes bacteria-seed interactions. The endophytic community of citrus seedlings changed according to treatment. In seedlings treated with the PR2/7 with pEglA clone, the population of group II decreased significantly, within the context of the total endophytic community. These results indicate that the application of GMEs induces shifts in the endophytic bacterial community of citrus seedlings.
- Characterization of Protocatechuate 4,5-Dioxygenase Induced from p-Hydroxybenzoate-Cultured Pseudomonas sp. K82
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Sung-Ho Yun , Chi-Young Yun , Seung Il Kim
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J. Microbiol. 2004;42(2):152-155.
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DOI: https://doi.org/2029 [pii]
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Pseudomonas sp. K82 has been reported to be an aniline-assimilating soil bacterium. However, this strain can use not only aniline as a sole carbon and energy source, but can also utilize benzoate, phydroxybenzoate, and aniline analogues. The strain accomplishes this metabolic diversity by using different aerobic pathways. Pseudomonas sp. K82, when cultured in p-hydroxybenzoate, showed extradiol cleavage activity of protocatechuate. In accordance with those findings, our study attempted the purification of protocatechuate 4,5-dioxygenase (PCD 4,5). However the purified PCD 4,5 was found to be very unstable during purification. After Q-sepharose chromatography was performed, the crude enzyme activity was augmented by a factor of approximately 4.7. From the Q-sepharose fraction which exhibited PCD 4,5 activity, two subunits of PCD4,5 ([alpha]subunit and [beta] subunit) were identified using the N-terminal amino acid sequences of 15 amino acid residues. These subunits were found to have more than 90% sequence homology with PmdA and PmdB of Comamonas testosteroni. The molecular weight of the native enzyme was estimated to be approximately 54 kDa, suggesting that PCD4,5 exists as a heterodimer ([alpha]_1[beta]_1). PCD 4,5 exhibits stringent substrate specificity for protocatechuate and its optimal activity occurs at pH 9 and 15^oC. PCR amplification of these two subunits of PCD4,5 revealed that the [alpha] subunit and [beta] subunit occurred in tandem. Our results suggest that Pseudomonas sp. K82 induced PCD 4,5 for the purpose of p-hydroxybenzoate degradation.
- Antibody Response to Crude Cell Lysate of Propionibacterium acnes and Induction of Pro-Inflammatory Cytokines in Patients with Acne and Normal Healthy Subjects
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Basal , E. , A. , Kaushal , G.P.
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J. Microbiol. 2004;42(2):117-125.
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DOI: https://doi.org/2035 [pii]
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Propionibacterium acnes (P. acnes) plays an important role in the disease pathogenesis of acne vulgaris, a disorder of pilosebaceous follicles, seen primarily in the adolescent age group. In the present study, the presence of antibodies against P. acnes (MTCC1951) were detected in acne patient (n=50) and disease free controls (n=25) using dot-ELISA and Western blot assay. The ability of P. acnes to induce proinflammatory cytokines by human peripheral blood mononuclear cells (PBMCs), obtained from acne patients and healthy subjects, were also analysed. The patients (n=26) who were culture positive for skin swab culture, were found to have a more advanced disease and higher antibody titres (1:4000 to >1:16000) compared to the P. acnes negative patients (n=24) and normal controls (n=25). An analysis of patients' sera by western blot assay recognized a number of antigenic components of P. acnes, ranging from 29 to 205 kDa. The major reactive component was an approximately 96 kDa polypeptide, which was recognised in 92% (24 of 26) of the patients sera. Further, the P. acnes culture supernatant, crude cell lysate and heat killed P. acnes whole cells, obtained from 72-h incubation culture, were observed to be able to induce significant amounts of IL-8 and tumor necrosis factor alpha (TNF-[alpha]) by the PBMCs in both the healthy subjects and patients, as analysed by cytokine-ELISA. The levels of cytokines were significantly higher in the patients than the healthy subjects. A major 96 kDa polypeptide reactant was eluted from the gel and was found to cause dose dependent stimulation of the productions of IL-8 and TNF-[alpha]. Thus, the above results suggest that both humoral and pro-inflammatory responses play major roles in the pathogenesis of acne.
- Growth Inhibition of Escherichia coli during Heterologous Expression of Bacillus subtilis Glutamyl-tRNA Synthetase that Catalyzes the Formation of Mischarged Glutamyl-tRNA_1^Gln
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Ji-Won Baick , Jang-Ho Yoon , Suk Namgoong , Dieter S?l , Sung-Il Kim
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J. Microbiol. 2004;42(2):111-116.
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DOI: https://doi.org/2036 [pii]
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It is known that Bacillus subtilis glutamyl-tRNA synthetase (GluRS) mischarges E. coli tRNA_1^Gln with glutamate in vitro. It has also been established that the expression of B. subtilis GluRS in Escherichia coli results in the death of the host cell. To ascertain whether E. coli growth inhibition caused by B. subtilis GluRS synthesis is a consequence of Glu-tRNA_1^ Gln formation, we constructed an in vivo test system, in which B. subtilis GluRS gene expression is controlled by IPTG. Such a system permits the investigation of factors affecting E. coli growth. Expression of E. coli glutaminyl-tRNA synthetase (GlnRS) also ameliorated growth inhibition, presumably by competitively preventing tRNA_1^Gln misacylation. However, when amounts of up to 10 mM L-glutamine, the cognate amino acid for acylation of tRNA_1^ Gln, were added to the growth medium, cell growth was unaffected. Overexpression of the B. subtilis gatCAB gene encoding GlutRNA^Gln amidotransferase (Glu-AdT) rescued cells from toxic effects caused by the formation of the mischarging GluRS. This result indicates that B. subtilis Glu-AdT recognizes the mischarged E. coli GlutRNA_1^Gln, and converts it to the cognate Gln-tRNA_1^Gln species. B. subtilis GluRS-dependent Glu-tRNA_1^Gln formation may cause growth inhibition in the transformed E. coli strain, possibly due to abnormal protein synthesis.
- Use of Sucrose-Agar Globule with Root Exudates for Mass Production of Vesicular Arbuscular Mycorrhizal Fungi
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Thangaswamy Selvaraj , Hoon Kim
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J. Microbiol. 2004;42(1):60-63.
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DOI: https://doi.org/1998 [pii]
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A sucrose-agar globule (SAG) was newly introduced to increase production of the vesicular arbuscular mycorrhizal (VAM) fungal spores, Gigaspora gigantea and Glomus fasciculatum. An SAG inoculum and a sucrose-agar globule with root exudates (SAGE) inoculum were prepared, and their spore productions were compared with a soil inoculum. When the SAGE was used as the inoculum on sucroseagar medium plates the number of spores was increased (35% more than the soil inoculum). After the soil inoculum and SAGE were inoculated on an experimental plant, Zingiber officinale, the percentage root colonization, number of VAM spores, and dry matter content were analyzed. It was observed that the SAGE showed a higher percentage of root colonization (about 10% more), and increases in the number of spores (about 26%) and dry matter (more than 13%) for the two VAM fungal spores than the soil inoculum. The results of this study suggested that the SAGE inoculum may be useful for the mass production of VAM fungi and also for the large scale production of VAM fungal fertilizer.
- Study on Persistent Infection of Japanese Encephalitis Virus Beijing-1 Strain in Serum-free Sf9 Cell Cultures
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Hun Kim , Su Jeen Lee , Jin Yong Park , Yong Wook Park , Hyun Sung Kim , Heui-Yun Kang , Byung-Ki Hur , Yeon-Woo Ryu , Sang In Han , Jong Su Kim
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J. Microbiol. 2004;42(1):25-31.
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DOI: https://doi.org/2005 [pii]
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Sf9 cells have obvious advantages for the conventional production technology of vaccine. They are useful tools for high concentration and large-scale cultures. Sf9 cells were grown to maximal concentration, 8×10^6 cells/ml in a 500ml spinner flask, with a doubling time at the exponentially growing phase of 24.5 hours, using serum-free media. To explore the ability of Sf9 cells to be infected by the Japanese encephalitis (JE) virus Beijing-1 strain, Sf9 cells were infected with the virus. By 4-5 days post-infection, 10-15% of the Sf9 cells showed cytopathic effect (CPE), from granularity to the formation of syncytia and multinucleated giant cells continuously observed over a period of 35 days. Positive fluorescent reactions were detected in 30-40% of cells infected with the JE virus Beijing-1 strain, and the uninfected Sf9 cells were completely negative. Virus particles, propagated in Sf9 and Vero cells, were concentrated by sedimentation on 40% trehalose cushions by ultracentrifugation, and showed identical patterns of viral morphogenesis. Complete virus particles, 40 to 50 nm in diameter, were observed, and JE virus envelope (E) proteins, at 53 kDa, were found in the western blot analysis to the anti-JE virus E protein monoclonal antibody and reacted as a magenta band in the same position to the glycoprotein staining. To evaluate whether the infectious virus was produced in Sf9 cells inoculated with the JE virus Beijing-1 stain, Sf9 cells were inoculated with the virus, and sample harvested every 5 days. The titers of the JE virus Beijing-1 strain rose from 1.0×10^5 to 1.5×10^6 pfu/ml. The infected Sf9 cells could be subcultured in serum-free medium, with no change in the plaque sizes formed by the JE virus Beijing-1 strain in the plaque assay. It is suggested that the ability of the JE virus Beijing-1 strain to infect Sf9 cells in serum-free media will provide a useful insect cell system, where the JE virus replication, cytopathogenicity and vaccine immunogen can be studied.
- Study on Persistent Infection of Japanese Encephalitis Virus Beijing-1 Strain in Serum-free Sf9 Cell Cultures
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Hun Kim , Su Jeen Lee , Jin Yong Park , Yong Wook Park , Hyun Sung Kim , Heui-Yun Kang , Byung-Ki Hur , Yeon-Woo Ryu , Sang In Han , Jong Su Kim
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J. Microbiol. 2004;42(1):25-31.
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DOI: https://doi.org/2005 [pii]
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Sf9 cells have obvious advantages for the conventional production technology of vaccine. They are useful tools for high concentration and large-scale cultures. Sf9 cells were grown to maximal concentration, 8×10^6 cells/ml in a 500ml spinner flask, with a doubling time at the exponentially growing phase of 24.5 hours, using serum-free media. To explore the ability of Sf9 cells to be infected by the Japanese encephalitis (JE) virus Beijing-1 strain, Sf9 cells were infected with the virus. By 4-5 days post-infection, 10-15% of the Sf9 cells showed cytopathic effect (CPE), from granularity to the formation of syncytia and multinucleated giant cells continuously observed over a period of 35 days. Positive fluorescent reactions were detected in 30-40% of cells infected with the JE virus Beijing-1 strain, and the uninfected Sf9 cells were completely negative. Virus particles, propagated in Sf9 and Vero cells, were concentrated by sedimentation on 40% trehalose cushions by ultracentrifugation, and showed identical patterns of viral morphogenesis. Complete virus particles, 40 to 50 nm in diameter, were observed, and JE virus envelope (E) proteins, at 53 kDa, were found in the western blot analysis to the anti-JE virus E protein monoclonal antibody and reacted as a magenta band in the same position to the glycoprotein staining. To evaluate whether the infectious virus was produced in Sf9 cells inoculated with the JE virus Beijing-1 stain, Sf9 cells were inoculated with the virus, and sample harvested every 5 days. The titers of the JE virus Beijing-1 strain rose from 1.0×10^5 to 1.5×10^6 pfu/ml. The infected Sf9 cells could be subcultured in serum-free medium, with no change in the plaque sizes formed by the JE virus Beijing-1 strain in the plaque assay. It is suggested that the ability of the JE virus Beijing-1 strain to infect Sf9 cells in serum-free media will provide a useful insect cell system, where the JE virus replication, cytopathogenicity and vaccine immunogen can be studied.
- Study on Persistent Infection of Japanese Encephalitis Virus Beijing-1 Strain in Serum-free Sf9 Cell Cultures
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Hun Kim , Su Jeen Lee , Jin Yong Park , Yong Wook Park , Hyun Sung Kim , Heui-Yun Kang , Byung-Ki Hur , Yeon-Woo Ryu , Sang In Han , Jong Su Kim
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J. Microbiol. 2004;42(1):25-31.
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DOI: https://doi.org/2005 [pii]
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Abstract
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Sf9 cells have obvious advantages for the conventional production technology of vaccine. They are useful tools for high concentration and large-scale cultures. Sf9 cells were grown to maximal concentration, 8×10^6 cells/ml in a 500ml spinner flask, with a doubling time at the exponentially growing phase of 24.5 hours, using serum-free media. To explore the ability of Sf9 cells to be infected by the Japanese encephalitis (JE) virus Beijing-1 strain, Sf9 cells were infected with the virus. By 4-5 days post-infection, 10-15% of the Sf9 cells showed cytopathic effect (CPE), from granularity to the formation of syncytia and multinucleated giant cells continuously observed over a period of 35 days. Positive fluorescent reactions were detected in 30-40% of cells infected with the JE virus Beijing-1 strain, and the uninfected Sf9 cells were completely negative. Virus particles, propagated in Sf9 and Vero cells, were concentrated by sedimentation on 40% trehalose cushions by ultracentrifugation, and showed identical patterns of viral morphogenesis. Complete virus particles, 40 to 50 nm in diameter, were observed, and JE virus envelope (E) proteins, at 53 kDa, were found in the western blot analysis to the anti-JE virus E protein monoclonal antibody and reacted as a magenta band in the same position to the glycoprotein staining. To evaluate whether the infectious virus was produced in Sf9 cells inoculated with the JE virus Beijing-1 stain, Sf9 cells were inoculated with the virus, and sample harvested every 5 days. The titers of the JE virus Beijing-1 strain rose from 1.0×10^5 to 1.5×10^6 pfu/ml. The infected Sf9 cells could be subcultured in serum-free medium, with no change in the plaque sizes formed by the JE virus Beijing-1 strain in the plaque assay. It is suggested that the ability of the JE virus Beijing-1 strain to infect Sf9 cells in serum-free media will provide a useful insect cell system, where the JE virus replication, cytopathogenicity and vaccine immunogen can be studied.
- A Method for Comparing Multiple Bacterial Community Structures from 16S rDNA Clone Library Sequences
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Inae Hur , Jongsik Chun
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J. Microbiol. 2004;42(1):9-13.
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DOI: https://doi.org/2008 [pii]
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Culture-independent approaches, based on 16S rDNA sequences, are extensively used in modern microbial ecology. Sequencing of the clone library generated from environmental DNA has advantages over fingerprint-based methods, such as denaturing gradient gel electrophoresis, as it provides precise identification and quantification of the phylotypes present in samples. However, to date, no method exists for comparing multiple bacterial community structures using clone library sequences. In this study, an automated method to achieve this has been developed, by applying pair wise alignment, hierarchical clustering and principle component analysis. The method has been demonstrated to be successful in comparing samples from various environments. The program, named CommCluster, was written in JAVA, and is now freely available, at http://chunlab.snu.ac.kr/commcluster/.
- Functional Implications in Apoptosis by Interferon Inducible Gene Product 1-8D, the Binding Protein to Adenovirus Preterminal Protein
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Insil Joung , Jeffrey A. Engler
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J. Microbiol. 2003;41(4):295-299.
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Adenovirus (Ad) precursor to the terminal protein (pTP) plays an essential roles in the viral DNA replication. Ad pTP serves as a primer for the synthesis of a new DNA strand during the initiation step of replication. In addition, Ad pTP forms organized spherical replication foci on the nuclear matrix (NM) and anchors the viral genome to the NM. Here we identified the interferon inducible gene product 1-8D (Inid) as a pTP binding protein by using a two-hybrid screen of a HeLa cDNA library. Of the clones obtained in this assay, nine were identical to the Inid, a 13-kDa polypeptide that shares homology with genes 1-8U and Leu-13/9-27, most of which have little known functions. The entire open reading frame (ORF) of Inid was cloned into the tetracycline inducible expression vector in order to determine the biological functions related with adenoviral infection. When Inid was introduced to the cells along with adenoviruses, fifty to sixty percent of Ad-infected cells expressing Inid had rounded morphology, which was suggestive of apoptosis. Results from the terminal deoxynucleotidyl transferase (TdT) and DNA fragmentation assays confirmed that Inid induces apoptosis in Ad-infected or in uninfected cells. The Inid binding to pTP may target the cell for apoptotic destruction as a host defense mechanism against the viral infection.
- Characterization of the Proteolytic Activity of Bacteria Isolated from a Rotating Biological Contactor
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In Jae Park , Jerng Chang Yoon , Seong Joo Park , Eung Ho Kim , Yeon-Jae Cho , Kwang-Soo Shin
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J. Microbiol. 2003;41(2):73-77.
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Four proteolytic bacteria were isolated and identified from a rotating biological contactor based on Bacillus. The four isolates, Ni 26, 36, 39 and 49 were identified as B. vallismortis, B. subtilis, Aeromonas hydrophila and B. amyloliquefaciens, respectively, based on their biochemical properties and 16S rDNA sequence analyses. The optimal proteolytic activity was observed in the temperature and pH ranges of 40-70ºC and 8.0-8.5, respectively. The proteolytic activities of all the isolates were partially inhibited by phenylmethylsulfonylfluoride (PMSF), and the isolates Ni 26, Ni 39 and Ni 49 were inhibited by the metalloprotease inhibitor, 1,10-phenanthroline. Zymographic analyses of the culture supernatants revealed the presence of at least two proteases in all isolates.
- Tsukamurella sunchonensis sp. nov., a Bacterium Associated with Foam in Activated Sludge
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Chi Nam Seong , Young Sook Kim , Keun Shik Baik , Sang Ki Choi , Min Bae Kim
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J. Microbiol. 2003;41(2):83-88.
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aeration basin of an activated sludge process, was clarified by phenotypic, chemotaxonomic and phylogenetic analyses. The strain possesses wall chemotype IV, MK-9(H_0 ), as the major menaquinone, and contains saturated, monounsaturated and 10-methyl branched fatty acids. The G+C content of its DNA is 68.1 mol%. Phenotypic data and DNA relatedness to known species indicate that the strain SCNU5^T represents a new species within the genus Tsukamurella, for which we propose the name Tsukamurella sunchonensis sp. nov. The type strain of T. sunchonensis is SCNU5^T (=KCTC 9827^T ).
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